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Method of modulating the activity of calcium channels in cardiac cells and reagents therefor

a technology of calcium channel and activity, which is applied in the direction of peptide/protein ingredients, peptide sources, instruments, etc., can solve the problems of myocardial contractile failure, morbidity and mortality, and contractile failur

Inactive Publication Date: 2005-03-03
AUSTRALIEN NAT UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0023] It will be apparent from the preceding discussion that one embodiment of the invention is directed to a method for enhancing the activity of a cardiac RyR2 calcium channel comprising contacting a cardiac RyR2 with an amount of a fragment of a dihydropyridine receptor (DHPR) polypeptide sufficient to enhance the activity of said RyR2, and determining the activity of said cardiac RyR2 calcium channel. As exemplified herein and without limiting the invention to any theory or mode of action or effective peptide concentration, the present inventors have shown that, for isolated cardiac RyR2 in a lipid bilayer, both the frequency of channel openings and the duration of each channel opening is enhanced by the application of up to about 1 nM peptide to about 10 .mu.M peptide. At high peptide concentrations, the activity of channels that opened first declines slightly however the activity of other channels is more pronounced, presumably reflecting a microheterogeneity in RyR2 channel sensitivities to peptide.

Problems solved by technology

Myocardial contractile failure is a common cause of morbidity and mortality in patients with ischemic heart disease, congestive heart failure, and systemic inflammatory states such as sepsis.
Accumulating evidence indicates that contractile failure is associated with dysregulation of myoplasmic calcium flux.
However, individual DHPRs and RyRs in failing or hypertrophic hearts appear normal, suggesting that the link between these two calcium signal proteins may be defective.
Additionally, hyperphosphorylation of RyR2 in failing human hearts results in defective channel function.
Discrete areas of heart muscle are damaged during ischaemic episodes, causing reduced cardiac output.
In the long term increased cAMP levels can become toxic and lead to calcium overload.

Method used

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  • Method of modulating the activity of calcium channels in cardiac cells and reagents therefor
  • Method of modulating the activity of calcium channels in cardiac cells and reagents therefor
  • Method of modulating the activity of calcium channels in cardiac cells and reagents therefor

Examples

Experimental program
Comparison scheme
Effect test

example 2

A 20-Mer Peptide that Modulates Cardiac RyR2 Calcium Channel Activity

Results

[0174] An increase in the activity of RyRs from cardiac muscle was seen when the 20-mer peptide (SEQ ID NO: 2) was added to the cytoplasmic (cis) side of the channel at a concentration of 10.sup.-7 cis Ca.sup.2+. Single channels were identified as RyRs by their Cs.sup.+ conductance of .about.450 pS with bilayer potentials of +40 mV or -40 mV and by the ability of 30 .mu.M ruthenium red to block the channel at the end of the experiment.

[0175] Records from one experiment in which cardiac RyRs were strongly activated by the 20-mer peptide (SEQ ID NO: 2) are shown in FIG. 2, at -40 mV, and in FIG. 3, at +40 mV. Channel activity before addition of the peptide consisted of brief intermittent openings (FIG. 2, panel A; FIG. 3, panel A). Within 10 s of adding 65 nM peptide (SEQ ID NO: 2), channel openings increased at -40 mV.

[0176] An increase in the frequency of events, and the appearance of very long openings, wer...

example 3

Functional Analysis of DHPR 20-Mer Fragment Derivatives and Analogues

Materials & Methods

[0185] Peptides

[0186] Four peptides were tested in this series of experiments, these being:

[0187] (i) the native DHPR 20-mer peptide (SEQ ID NO: 2);

[0188] (ii) the SEQ ID NO: 2 peptide in which Ser.sup.687 (residue 17) is replaced by an alanine residue to produce SEQ ID NO: 8;

[0189] (iii) the SEQ ID NO: 2 peptide in which Arg.sup.688 (residue 18) is replaced by an D isomer to produce SEQ ID NO: 9; and

[0190] (iv) the SEQ ID NO: 2 peptide in which Ser.sup.687 is mutated to alanine and Arg.sup.688 is replaced by a D isomer to produce SEQ ID NO: 10.

[0191] Measurements of Ca.sup.2+ Release from Cardiac SR

[0192] Cardiac SR vesicles (50 .mu.g of protein) were added to a cuvette, to a final volume of 2 ml of a solution containing (in mM): 100, KH.sub.2PO.sub.4 (pH=7); 4, MgCl.sub.2; 1, Na.sub.2ATP; 0.5, antipyrylazo III. Extravesicular [Ca.sup.2+] was monitored at 710 nm using either a Cary 50 or Cary 10...

example 4

Functional Analysis of DHPR 20-Mer Peptide Fragment Derivatives and Analogues

Results

[0195] Summary

[0196] The example 4 experiments include the testing of all compounds on Ca.sup.2+ release from cardiac SR vesicles. In addition, single cardiac RyR channels were exposed to peptide SEQ ID NO: 9. All peptides significantly enhanced the rates of Ca.sup.+-activated Ca.sup.2+ release or caffeine-activated Ca.sup.2+ release. Peptides SEQ ID NOs: 9 and 10, at high concentrations of >30 .mu.M marginally enhanced the rate of Ca.sup.2+ release in the absence of activating Ca.sup.2+ or caffeine, and also enhanced both Ca.sup.2+-activated Ca.sup.2+ release and caffeine-activated Ca.sup.2+ release. Peptide SEQ ID NO: 9 enhanced cardiac RyR activity in lipid bilayer experiments at low concentrations (1-10 nM), but blocked the channel pore in a voltage- and Ca.sup.2+-dependent manner at +40 mV at high concentrations. Accordingly, under appropriate physiological conditions, the dihydropyridine recept...

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Abstract

The present invention relates generally to novel peptides that are capable of modulating the activity of calcium channels in cardiac cells. More specifically, the present invention provides a method of modulating the activity of a cardiac calcium channel comprising contacting a cardiac ryanodine receptor (RyR2) with an amount of a fragment of a dihydropyridine receptor (DHPR) polypeptide sufficient to modulate the activity of said RyR2, and determining the activity of said calcium channel. The inventive method is useful for the treatment of a range of disorders and diseases associated with cardiac dysfunction, particularly those diseases and disorders involving reduced cardiac output and / or aberrant excitation-contraction coupling, calcium overload, or calcium leakage, in cardiac cells.

Description

[0001] The present invention relates generally to novel peptides that are capable of modulating the activity of calcium channels in cardiac cells. More specifically, the present invention provides a method of modulating the activity of a cardiac calcium channel comprising contacting a cardiac ryanodine receptor (RyR2) with an amount of a fragment of a dihydropyridine receptor (DHPR) polypeptide sufficient to modulate the activity of said RyR2, and determining the activity of said calcium channel. The inventive method is useful for the treatment of a range of disorders and diseases associated with cardiac dysfunction, particularly those diseases and disorders involving reduced cardiac output and / or aberrant excitation-contraction coupling, calcium overload, or calcium leakage, in cardiac cells.BACKGROUND TO THE INVENTION[0002] Bibliographic details of the publications referred to in this specification are collected at the end of the description. Reference herein to prior art, includi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00G01N33/50A61K38/17A61P9/00A61P9/04A61P9/06A61P9/10A61P29/00A61P31/04A61P43/00C07K14/435C07K14/705G01N33/15G01N33/566G01N33/68G01N33/94
CPCC07K14/705G01N33/566G01N33/6893A61K38/00G01N2500/00G01N2800/32G01N33/9453A61P29/00A61P31/04A61P43/00A61P9/00A61P9/04A61P9/06A61P9/10A61K38/16
Inventor DULHUNTY, ANGELA F.CASAROTTO, MARCO G.
Owner AUSTRALIEN NAT UNIV
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